Project Summary. With the majority of therapeutic drugs available on the market being natural products or derivatives of them, understanding how organisms and enzymes function to produce these structurally complex compounds is essential. Polyketides are a class of secondary metabolites that are biosynthesized by polyketide synthases (PKSs) and often serve as antibacterial, antifungal, and anticancer agents. The PKSs are complex biological machineries that involve proteins and substrates interacting with one another with high specificity to assemble polyketides. These unique protein-protein and protein-substrate interactions are the basis for how these synthases are governed and are therefore critical to understand. Common in all three types of PKSs is the iterative elongation of polyketide intermediates by two-carbon units, but how their respective elongation enzymes function and stabilize the substrates while preventing them from undergoing unwanted side reactions continues to remain unknown. In this proposal, we aim to first understand the fit of growing polyketones in the pocket of a carrier protein-guided elongation enzyme by developing isosteric mimetics of polyketide intermediates from a type II PKS model. Here, we will use crosslinking to trap the partner proteins to elucidate the key interactions as the intermediates are elongated. We then plan to apply similar chemical biology tools in our second research aim to define the substrate interactions catalyzed by a CoA-dependent elongation enzyme in a type III PKS system. In this study, we will develop polyketide intermediate mimetics and malonyl-CoA analogs to be able to provide a snapshot of the natural substrate interactions through x-ray crystallography. These studies will allow us to uncover the molecular details that drive the elongation process responsible for building the core carbon backbone of polyketides. Gaining a deeper understanding of these protein and substrate interactions enables their manipulation and redesign to produce novel polyketides with different pharmacophores.